Carrier molecules, such as whole antibody molecules or the F(ab').sub.2, Fab' or Fab fragments thereof, have been used in conjugation with a broad spectrum of pharmaceutical or cytotoxic agents, such as radioactive compounds, for the purpose of the targeted delivery of the therapeutic agents to tumor cells. The therapeutic agents are preferably covalently attached to the antibody protein in a fashion that leaves the antibody unchanged with respect to its ability to bind antigen, (see, for example, U.S. Pat. No. 4,671,958 and the references therein).
Among the common ways of performing this chemical attachment is the use of the carbohydrate groups which are present on whole antibody molecules (J. Immunological Methods, 99, 153 (1985)). This method involves the oxidation of the carbohydrate groups to aldehydes, followed by reductive amination using an amine and a reducing agent. Unfortunately, this approach is not applicable with most Fab' antibody fragments from which the carbohydrate-bearing Fc region has typically been enzymatically cleaved.
A similar method (U.S. Pat. No. 4,093,607) describes the periodate oxidation of a therapeutic agent that contains a glycol or hydroxy group, thus forming an aldehyde which is subsequently reacted with an antibody molecule or fragment. Attachment occurs via the formation of a Schiff base with the amino groups of the antibody. The Schiff base linkage is subsequently reduced.
Bifunctional cross-linking agents, including maleimide and bismaleimide compounds, have been used to attach Fab' fragments to other protein and non-protein species through free Fab' sulfhydryl groups, again with retention of antigen binding activity (see e.g. U.S. Pat. No. 4,659,839; Biochem. Biophys. Res. Commun., 99, 1146 (1981); and J. Biol. Chem., 257, 286 (1982)). Recently, bismaleimides have been used to prepare bispecific F(ab').sub.2 species containing thioether-linked Fab' fragments (J. Immunology, 139, 2367 (1987)). Heretofore, however, antibody fragments or synthetically recombined antibody fragments having a linking moiety containing an aldehyde or masked aldehyde functionality suitable for attachment of a therapeutic agent bearing a reactive amino functionality have been unknown.